SYN3 compositions and methods

ABSTRACT

Disclosed are aqueous and nonaqueous solution formulations containing agents that are useful for treating cancer.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. patent application Ser.No. 60/342,329, filed Dec. 20, 2001, the disclosure of which is herebyincorporated by reference in its entirety for all purposes.

FIELD OF INVENTION

[0002] The present invention is directed to compositions for treatingcancer by gene therapy using a therapeutic gene, such as a tumorsuppressor gene delivered by a gene delivery system, such as arecombinant viral vector delivery system, in combination with atransduction enhancing agent. In particular, this invention relates tothe delivery of a tumor suppressor gene (e.g., p53 or retinoblastoma(RB)) to cancerous epithelial tissues and organs, such as the bladder,using a recombinant adenoviral vector delivery system formulated in astabilized buffer in combination with a transduction enhancing agent,such as SYN3.

BACKGROUND OF THE INVENTION

[0003] Epithelial Cancer is an insidious disease. For instance, one typeof epithelial cancer, carcinoma of the bladder, represents a significantsource of morbidity and mortality. Bladder cancer reportedly ranks 10thin males and 12th in females in cancer related mortality. Therapiesavailable for the treatment of bladder cancer include adjuvantchemotherapy or immunotherapy, transurethral resection of superficialdisease, radical cystectomy or radiotherapy which is often combined withsystemic chemotherapy. Despite these therapeutic options, overallsurvival has not changed appreciably. Thus, new therapeutic modalitiesmust be developed for the treatment of bladder cancer.

[0004] Gene therapy strategies have been reportedly developed as analternative therapeutic approach. Distinct approaches have beendeveloped to treat neoplasms based on gene transfer methods. Methodshave been reportedly developed to correct specific lesions at definedgenetic loci which give rise to neoplastic transformation andprogression. Overexpression of dominant oncogenes can be addressed usingtechniques to inhibit the transforming gene or gene product. It has beenreported that loss of tumor suppressor gene function may be approachedusing methods to reconstitute wild-type tumor suppressor gene. Besidesthese methods to achieve mutation compensation, genetic techniques havebeen reportedly developed to specifically and selectively eradicatetumor cells. These approaches of molecular chemotherapy reportedly relyon specific expression of toxin genes in neoplastic cells. Finally, genetransfer methods have been reportedly used to achieve antitumorimmunization. These methods of genetic immunopotentiation reportedly usetechniques of genetic immunoregulation to enhance immune recognition oftumors. Consequently, a variety of distinct approaches reportedly havebeen developed to accomplish gene therapy of cancer.

[0005] A high incidence of mutations has reportedly been observed intumor suppressor genes, such as p53 and RB, in the case of carcinoma ofthe bladder. For such genetic lesions of tumor suppressor genes,reversion of the neoplastic phenotype can be demonstrated withreplacement of the corresponding wild-type tumor suppressor gene.

[0006] In vitro studies using cell lines derived from human bladdertissues have reportedly demonstrated efficient transgene expressionfollowing infection with recombinant adenovirus. Experiments in vivoreportedly have also shown adenovirus transgene expression in theurinary bladder of rodents after intravesical delivery. In vitroexperiments with wild-type adenovirus demonstrate that virus attachmentand internalization is not influenced by benzyl alcohol, but doreportedly demonstrate an enhanced uncoating of the virion. In vivoefforts with agents (e.g. acetone, DMSO, prolamine sulfate) canreportedly break down the protective “mucin” layer that protects thebladder epithelium from bacteria, viruses and other pathogens.

[0007] U.S. Pat. No. 5,789,244 claims a composition comprising a viralvector in which a nucleotide sequence encoding a transgene has beeninserted, wherein the viral vector is formulated in a buffer comprisingethanol in a concentration range of about 1% to 50% (v/v). U.S. Pat. No.5,837,520 claims a method for purification of an intact viral particlefrom a cell lysate comprising treating the cell lysate which containsthe intact viral particle with an enzymatic agent that selectivelydegrades both unencapsulated DNA and RNA; chromatographing the treatedlysate from the first step on a first resin; and chromatographing theeluant from the second step on a second resin; wherein one resin is ananion exchange resin and the other is an immobilized metal ion affinityresin. U.S. Pat. No. 5,932,210 describes a composition comprising arecombinant adenovirus expression vector and a pharmaceuticallyacceptable carrier, the vector comprising: (a) an insert of exogenousDNA comprising a gene encoding a foreign protein; and (b) adenovirus DNAin which all of the coding sequences of E1a, E1b, and protein IX, and atleast part of E3 have been deleted. U.S. Pat. No. 6,165,779 discloses acomposition comprising a recombinant virus vector formulated in a buffercomprising a detergent. U.S. Pat. No. 6,210,939 claims a recombinantadenovirus expression vector comprising (a) an insert of exogenous DNAcomprising a gene encoding a foreign protein and (b) adenovirus DNAwhich has sustained a deletion beginning at nucleotide 357 and ending atnucleotide 4020 to 4050. Finally, U.S. Pat. No. 6,312,681 discloses amethod for delivering an adenoviral vector which comprises a transgeneto a cancer cell in the epithehial membrane of a bladder, the methodcomprising administering to the epithelial membrane the adenoviralvector and between 1% and 50% (v/v) ethanol, wherein the adenoviralvector infects the cell and the transgene is expressed in infectedcells. All of these references are hereby incorporated by referencethereto in their entirety.

[0008] Notwithstanding the foregoing, there exists a need forformulations for therapeutic use that improve the efficiency of thetransgene delivery. Vectors that are unstable present difficulty inadministering the desired therapeutic agent to the patient. Because ofin vivo instability, there is a need for vector stabilization such thatthere is an increase in the transduction of the therapeutic agent thatis to be administered.

SUMMARY OF THE INVENTION

[0009] Accordingly, in one aspect, the present invention provides apharmaceutical composition comprising SYN3 in combination with apharmaceutically acceptable carrier.

[0010] A further aspect of the invention is a pharmaceutical compositioncomprising SYN3 in combination with a pharmaceutically acceptablenonaqueous carrier.

[0011] A further aspect of the invention is a pharmaceutical compositioncomprising SYN3 in combination with a pharmaceutically acceptableaqueous carrier.

[0012] A further aspect of the invention is a pharmaceutical compositioncomprising SYN3 in combination with a pharmaceutically acceptablecarrier and at least one pharmaceutically acceptable solubilizer.

[0013] A further aspect of the invention is a pharmaceutical compositioncomprising SYN3 in combination with a pharmaceutically acceptablenonaqueous carrier and at least one pharmaceutically acceptablesolubilizer.

[0014] A further aspect of the invention is a pharmaceutical compositioncomprising SYN3 in combination with a pharmaceutically acceptableaqueous carrier and at least one pharmaceutically acceptablesolubilizer.

[0015] A further aspect of the invention is a lyophilized pharmaceuticalcomposition comprising SYN3 in combination with a pharmaceuticallyacceptable carrier, at least one pharmaceutically acceptable solubilizerand a at least one pharmaceutically acceptable bulking agent.

[0016] A further aspect of the invention is a pharmaceutical compositioncomprising SYN3 in combination with a pharmaceutically acceptableaqueous carrier, at least one pharmaceutically acceptable solubilizer,and at least one pharmaceutically acceptable bulking agent.

[0017] A further aspect of the invention is a pharmaceutical compositioncomprising SYN3 in combination with a pharmaceutically acceptableaqueous carrier, at least one pharmaceutically acceptable solubilizer,at least one pharmaceutically acceptable bulking agent and at least onepharmaceutically acceptable buffering agent.

[0018] A further aspect of the invention is a pharmaceutical compositioncomprising SYN3 in combination with a pharmaceutically acceptablecarrier and an expression vector comprising a foreign DNA sequenceinserted into the vector.

[0019] A further aspect of the invention is a pharmaceutical compositioncomprising SYN3 in combination with a pharmaceutically acceptablenonaqueous carrier and an expression vector comprising a foreign DNAsequence inserted into the vector.

[0020] A further aspect of the invention is a pharmaceutical compositioncomprising SYN3 in combination with a pharmaceutically acceptableaqueous carrier and an expression vector comprising a foreign DNAsequence inserted into the vector.

[0021] A further aspect of the invention is a pharmaceutical compositioncomprising SYN3 in combination with a pharmaceutically acceptablecarrier, an expression vector comprising a foreign DNA sequence insertedinto the vector and at least one pharmaceutically acceptablesolubilizer.

[0022] A further aspect of the invention is a pharmaceutical compositioncomprising SYN3 in combination with a pharmaceutically acceptablenonaqueous carrier, an expression vector comprising a foreign DNAsequence inserted into the vector and at least one pharmaceuticallyacceptable solubilizer.

[0023] A further aspect of the invention is a pharmaceutical compositioncomprising SYN3 in combination with a pharmaceutically acceptableaqueous carrier, an expression vector comprising a foreign DNA sequenceinserted into the vector and at least one pharmaceutically acceptablesolubilizer.

[0024] A further aspect of the invention is a pharmaceutical compositioncomprising SYN3 in combination with a pharmaceutically acceptableaqueous carrier, an expression vector comprising a foreign DNA sequenceinserted into the vector, at least one pharmaceutically acceptablesolubilizer, and at least one pharmaceutically acceptable bulking agent.

[0025] A further aspect of the invention is a pharmaceutical compositioncomprising SYN3 in combination with a pharmaceutically acceptableaqueous carrier, an expression vector comprising a foreign DNA sequenceinserted into the vector, at least one pharmaceutically acceptablesolubilizer, at least one pharmaceutically acceptable bulking agent andat least one pharmaceutically acceptable buffering agent.

[0026] A further aspect of the invention is a use of SYN3 in thepreparation of a medicament for the treatment of bladder cancer.

[0027] A further aspect of the invention is a method of treating adisease in a mammal comprising administering a therapeutically effectiveamount of a pharmaceutical composition comprising SYN3 in combinationwith a pharmaceutically acceptable carrier.

[0028] These and other aspects, objects and advantages will become moreapparent when read with the accompanied detailed description whichfollows.

DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1 illustrates a chemical structural formula of SYN3.

[0030]FIG. 2 illustrates one method for the synthesis of SYN3.

DETAILED DESCRIPTION OF THE INVENTION

[0031] Accordingly, one aspect of the invention is that a uniquesurfactant-like molecule SYN3 is formulated with excipients to maintainsolubility and stability as well as compatibility with the adenovirus.

[0032] A further aspect of the invention is that the SYN3 formulationsare nontoxic to tissues, e.g., the bladder with which it comes incontact at therapeutic levels. Indeed, surfactants which act aspermeation enhancers often produce some toxicity due to membraneirritation. The use of SYN3 thus provides this further benefit ofavoiding this toxicity. Connor, et al., Gene Therapy, Vol. 8, pp. 41-48(2001).

[0033] A further aspect of the invention is that the stability of thevector is unaffected by combination with the SYN3. Often, surfactantlevels required to improved transduction may impart instability to thevector. Combination of the adenovirus and SYN3 preparations produces amore potent admixture compared with adenovirus.

[0034] Buffered and lyophilized aqueous formulations as well asnonaqueous solution formulations of SYN3 were produced. SYN3, is(N-(3-cholamidopropyl)-N-(3 (actobionamidopropyl))—cholamide (FIG. 1).As will be apparent to those of skill in the art, SYN3 exists in variousoptical, tautomeric, stereoisomeric and isomeric forms. FIG. 1illustrates a preferred isomer. However, the compositions of the presentinvention encompass all such forms in any percentage or racemic mixturethereof.

[0035] SYN3 is a surfactant-like molecule that enhances transduction ofrecombinant adenovirus/therapeutic gene vectors for treatment ofepithelial tissue and tumors, or, more specifically, in bladder tumors.SYN3 can be present in a concentration of from about 0.001 mg/ml toabout 150 mg/ml.

[0036] The term “therapeutic transgene” refers to a nucleotide sequencethe expression of which in the target cell produces a therapeuticeffect. The term therapeutic transgene includes but is not limited totumor suppressor genes, antigenic genes, cytotoxic genes, cytostaticgenes, pro-drug activating genes, apoptotic genes, pharmaceutical genesor anti-angiogenic genes. The vectors of the present invention may beused to produce one or more therapeutic transgenes, either in tandemthrough the use of IRES elements or through independently regulatedpromoters.

[0037] The term “tumor suppressor gene” refers to a nucleotide sequence,the expression of which in the target cell is capable of suppressing theneoplastic phenotype and/or inducing apoptosis. Examples of tumorsuppressor genes useful in the practice of the present invention includethe p53 gene, the APC gene, the DPC-4 gene, the BRCA-1 gene, the BRCA-2gene, the WT-1 gene, the retinoblastoma gene (Lee, et al., Nature,329:642 (1987)), the MMAC-1 gene, the adenomatous polyposis coli protein(Albertsen, et al., U.S. Pat. No. 5,783,666 issued Jul. 21, 1998), thedeleted in colon carcinoma (DCC) gene, the MMSC-2 gene, the NF-1 gene,nasopharyngeal carcinoma tumor suppressor gene that maps at chromosome3p21.3. (Cheng, et al., Proc. Nat. Acad. Sci., 95:3042-3047 (1998)), theMTS1 gene, the CDK4 gene, the NF-1 gene, the NF2 gene, and the VHL gene.

[0038] The term “antigenic genes” refers to a nucleotide sequence, theexpression of which in the target cells results in the production of acell surface antigenic protein capable of recognition by the immunesystem. Examples of antigenic genes include carcinoembryonic antigen(CEA), p53 (as described in Levine, A. PCT International Publication No.WO94/02167 published Feb. 3, 1994). In order to facilitate immunerecognition, the antigenic gene may be fused to the MHC class I antigen.

[0039] The term “cytotoxic gene” refers to nucleotide sequence, theexpression of which in a cell produces a toxic effect. Examples of suchcytotoxic genes include nucleotide sequences encoding pseudomonasexotoxin, ricin toxin, diptheria toxin, and the like.

[0040] The term “cytostatic gene” refers to nucleotide sequence, theexpression of which in a cell produces an arrest in the cell cycle.Examples of such cytostatic genes include p21, the retinoblastoma gene,the E2F-Rb gene, genes encoding cyclin dependent kinase inhibitors suchas P16, p15, p18 and p19, the growth arrest specific homeobox (GAX) geneas described in Branellec, et al. (PCT Publication WO97/16459 publishedMay 9, 1997 and PCT Publication WO96/30385 published Oct. 3, 1996).

[0041] The term “cytokine gene” refers to a nucleotide sequence, theexpression of which in a cell produces a cytokine. Examples of suchcytokines include GM-CSF, the interleukins, especially IL-1, IL-2, IL-4,IL-12, IL-10, IL-19, IL-20, interferons of the alpha, beta and gammasubtypes especially interferon α-2b and fusions such as interferonα-2α-1.

[0042] The term “chemokine gene” refers to a nucleotide sequence, theexpression of which in a cell produces a cytokine. The term chemokinerefers to a group of structurally related low-molecular cytokines weightfactors secreted by cells are structurally related having mitogenic,chemotactic or inflammatory activities. They are primarily cationicproteins of 70 to 100 amino acid residues that share four conservedcysteine. These proteins can be sorted into two groups based on thespacing of the two amino-terminal cysteines. In the first group, the twocysteines are separated by a single residue (C-x-C), while in the secondgroup, they are adjacent (C—C). Examples of member of the ‘C-x-C’chemokines include but are not limited to platelet factor 4 (PF4),platelet basic protein (PBP), interleukin-8 (IL-8), melanoma growthstimulatory activity protein (MGSA), macrophage inflammatory protein 2(MIP-2), mouse Mig (ml 19), chicken 9E3 (or pCEF-4), pig alveolarmacrophage chemotactic factors I and I (AMCF-I and -II), pre-B cellgrowth stimulating factor (PBSF),and IP10. Examples of members of the‘C—C’ group include, but are not limited to, monocyte chemotacticprotein 1 (MCP-1), monocyte chemotactic protein 2 (MCP-2), monocytechemotactic protein 3 (MCP-3), monocyte chemotactic protein 4 (MCP-4),macrophage inflammatory protein 1 alpha (MIP-1-alpha), macrophageinflammatory protein 1 beta (MIP-1-beta), macrophage inflammatoryprotein I gamma (MIP-1-gamma), macrophage inflammatory protein 3 alpha(MIP-3-alpha, macrophage inflammatory protein 3 beta (MIP-3-beta),chemokine (ELC), macrophage inflammatory protein 4 (MIP-4), macrophageinflammatory protein 5 (MIP-5), LD78 beta, RANTES, SIS-epsilon (p500),thymus and activation-regulated chemokine (TARC), eotaxin, 1-309, humanprotein HCC-1/NCC-2, human protein HCC-3, mouse protein C10.

[0043] The term “pharmaceutical protein gene” refers to nucleotidesequence, the expression of which results in the production of proteinhave pharmaceutically effect in the target cell. Examples of suchpharmaceutical genes include the proinsulin gene and analogs (asdescribed in PCT International Patent Application No. WO98/31397, growthhormone gene, dopamine, serotonin, epidermal growth factor, GABA, ACTH,NGF, VEGF (to increase blood perfusion to target tissue, induceangiogenesis, PCT publication WO98/32859 published Jul. 30, 1998),thrombospondin etc.

[0044] The term “pro-apoptotic gene” refers to a nucleotide sequence,the expression thereof results in the programmed cell death of the cell.Examples of pro-apoptotic genes include p53, adenovirus E3-11.6K, theadenovirus E4 or f4 gene, p53 pathway genes, and genes encoding thecaspases.

[0045] The term “pro-drug activating genes” refers to nucleotidesequences, the expression of which, results in the production of proteincapable of converting a nontherapeutic compound into a therapeuticcompound, which renders the cell susceptible to killing by externalfactors or causes a toxic condition in the cell. An example of a prodrugactivating gene is the cytosine deaminase gene. Cytosine deaminaseconverts 5-fluorocytosine to 5 fluorouracil, a potent antitumor agent).The lysis of the tumor cell provides a localized burst of cytosinedeaminase capable of converting 5FC to 5FU at the localized point of thetumor resulting in the killing of many surrounding tumor cells. Thisresults in the killing of a large number of tumor cells without thenecessity of infecting these cells with an adenovirus (the so-calledbystander effect”). Additionally, the thymidine kinase (TK) gene (seee.g. Woo, et al. U.S. Pat. No. 5,631,236 issued May 20, 1997 andFreeman, et al. U.S. Pat. No. 5,601,818 issued Feb. 11, 1997) in whichthe cells expressing the TK gene product are susceptible to selectivekilling by the administration of gancyclovir may be employed.

[0046] The term “anti-angiogenic” genes refers to a nucleotide sequence,the expression of which results in the extracellular secretion ofanti-angiogenic factors. Anti-angiogenesis factors include angiostatin,inhibitors of vascular endothelial growth factor (VEGF) such as Tie 2(as described in PNAS(USA)(1998) 95:8795-8800), endostatin.

[0047] It will be readily apparent to those of skill in the art thatmodifications and or deletions to the above referenced genes so as toencode functional subfragments of the wild type protein may be readilyadapted for use in the practice of the present invention. For example,the reference to the p53 gene includes not only the wild type proteinbut also modified p53 proteins. Examples of such modified p53 proteinsinclude modifications to p53 to increase nuclear retention as describedin Wahl, et al., Nat. Cell Biol., 3(12):E277-86 (2001), deletions suchas the delta13-19 amino acids to eliminate the calpain consensuscleavage site, modifications to the oligomerization domains (asdescribed in Bracco, et al. PCT published application WO97/0492 or U.S.Pat. No. 5,573,925).

[0048] It will be readily apparent to those of skill in the art that theabove therapeutic genes may be secreted into the media or localized toparticular intracellular locations by inclusion of a targeting moietysuch as a signal peptide or nuclear localization signal(NLS). Alsoincluded in the definition of therapeutic transgene are fusion proteinsof the therapeutic transgene with the herpes simplex virus type 1(HSV-1) structural protein, VP22. Fusion proteins containing the VP22signal, when synthesized in an infected cell, are exported out of theinfected cell and efficiently enter surrounding noninfected cells to adiameter of approximately 16 cells wide. This system is particularlyuseful in conjunction with transciptionally active proteins (e.g. p53)as the fusion proteins are efficiently transported to the nuclei of thesurrounding cells. See, e.g. Elliott, G. & O'Hare, P. Cell., 88:223-233(1997); Marshall, A. & Castellino, A. Research News Briefs., NatureBiotechnology, 15:205 (1997); O'Hare, et al. PCT publication WO97/05265published Feb. 13, 1997. A similar targeting moiety derived from the HIVTat protein is also described in Vives, et al., J. Biol. Chem.,272:16010-16017 (1997).

[0049] As used herein, “a gene delivery system” refers to any means ofdelivery of a therapeutic gene to a particular epithelial tissue ororgan including, for example, recombinant viral vectors and nonviralvector systems. Examples of nonvector systems include, but are notlimited to, any lipid-based, lipid encapsulated DNA or cationiclipid/DNA complexes. Examples of recombinant viral vectors include, butare not limited to, herpes virus, retrovirus, vaccinia virus,adenovirus, and adenoassociated viruses.

[0050] “Recombinant” refers to nucleic acids and protein encoded by themwherein the nucleic acids are constructed by methods of recombinant DNAtechnology, also termed “genetic engineering”. A preferred recombinantviral vector is the adenoviral vector delivery system which has adeletion of the protein IX gene. See International patent Application WO95/11984, which is herein incorporated by reference in its entirety. Therecombinant vector delivery system comprising a therapeutic gene, suchas a tumor suppressor gene, is formulated in a buffer that stabilizesthe vector and is combined with a delivery enhancing agent that iscompatible with the vector.

[0051] A “delivery-enhancing agent” refers to any agent which enhancesdelivery of a therapeutic gene, such as a tumor suppressor gene to acancerous tissue or organ. Such enhanced delivery may be achieved byvarious mechanisms. One such mechanism may involve the disruption of theprotective glycosaminoglycan layer on the epithelial surface of thebladder.

[0052] Examples of such delivery-enhancing agents are detergents,alcohols, glycols, surfactants, bile salts, heparin antagonists,cyclooxygenase inhibitors, hypertonic salt solutions, and acetates.Alcohols include, for example, the aliphatic alcohols such as ethanol,N-propanol, isopropanol, butyl alcohol, acetyl alcohol. Glycols include,for example, glycerol, propyleneglycol, polyethyleneglycol, andthioglycerol. Acetates such as acetic acid, gluconol acetate, and sodiumacetate are further examples of delivery-enhancing agents. Hypertonicsalt solutions like 1M NaCl are also examples of delivery-enhancingagents. Examples of surfactants are sodium dodecyl sulfate (SDS) andlysolecithin, polysorbate 80, nonylphenoxypolyoxyethylene,lysophosphatidylcholine, polyethylenglycol 400, polysorbate 20,polyoxyethylene ethers, and polyglycol ether surfactants. Bile saltssuch as taurocholate, sodium tauro-deoxycholate, deoxycholate,chenodesoxycholate, glycocholic acid, glycochenodeoxycholic acid andother astringents like silver nitrate may be used. Heparin-antagonistslike quaternary amines such as protamine sulfate may also be used.Cyclooxygenase inhibitors such as sodium salicylate, salicylic acid, andnonsteroidal antiinflammatory drug (NSAIDS) like indomethacin, naproxen,diclofenac may be used.

[0053] The term “enhanced” describes the increased delivery of thetherapeutic gene, such as a tumor suppressor gene, to the canceroustissue or organ. Increased delivery of a therapeutic gene, such as atumor suppressor gene, can be measured by various means, for example bymeasuring expression of the tumor suppressor gene compared to expressionlevels when the tumor suppressor gene is delivery in an adenoviralvector delivery system in a buffer lacking the delivery-enhancing agent.Examples of therapeutic genes are tumor suppressor genes and the suicidegene thymidine kinase. Examples of tumor suppressor genes include, butare not limited to, p53, the retinoblastoma gene, either full length,such as p110B, or fragments thereof such as p94RB or p56RB, Rb56, afunctional variant of Rb gene, a functional variant of the p53 gene, andp16. Other therapeutic genes include but are not limited to CFTR, genesencoding cytokines (such as the interferons alpha, beta, gamma, delta,interleukins (e.g., IL-4, IL-10, IL-2), GM-CSF, and any other genesencoding proteins which have therapeutic potential in the treatment ofnoncancerous diseases of the bladder such as cystitis. In someembodiments of the invention, the therapeutic gene encodes antisenseRNA.

[0054] In some embodiments, the compositions of the invention comprise atherapeutically effective amount of a therapeutic gene, such as a tumorsuppressor gene, contained in a recombinant viral vector delivery systemin a buffer comprising a delivery-enhancing agent. “Therapeuticallyeffective” as used herein refers to the prevention of, reduction of, orcuring of symptoms associated with a disease state.

[0055] Therapeutically effective amounts of the pharmaceuticalcomposition comprising a therapeutic gene, such as p53, or theretinoblastoma tumor suppressor gene, in a recombinant viral vectordelivery system formulated in a buffer comprising a delivery-enhancingagent will be administered in accord with the teaching of thisinvention. For example, therapeutically effective amounts of the p53tumor suppressor gene in the recombinant adenoviral vector deliverysystem formulated in a buffer containing a delivery-enhancing agent arein the range of about 1×10 particles/ml to 2×10¹² particles/ml, moretypically about 1×10⁸ particles/ml to 9×10¹¹ particles/ml, mosttypically 1×10¹⁰ particles/ml to 9×10¹¹ particles/ml.

[0056] P53, also known as ACN53, is a recombinant adenovirus type 5encoding wild-type p53 tumor suppressor protein, and is described in,for example, PCT patent application WO95/11984. In one aspect of theinvention, the formulated SYN3 is combined with p53 injection and theadmixture is instilled into the bladder. This preparation is intended totreat epithelial carcinomas. Preferably, p53 will be present in anamount of about 5 to about 8×10¹³ particles.

[0057] Detergents for use within the scope of the present inventioninclude, for example, anionic, cationic, zwitterionic, and nonionicdetergents. Exemplary detergents include, for example, but are notlimited to taurocholate, deoxycholate, taurodeoxycholate, cetylpyridium,benalkonium chloride, ZWITTERGENT 3-14 detergent, CHAPS(3-[(3-Cholamidopropyl)dimethylammoniol]-1-propanesulfonate hydrate,available from Aldrich, Big CHAP, Deoxy Big CHAP, TRITON-X-100 detergentavailable from Union Carbide, C12E8, Octyl-B-D-Glucopyranoside,PLURONIC-F64, PLURONIC-F68, PLURONIC-F69 detergents available form BASF,TWEEN20 detergent, and TWEEN80 detergent available from ICI.

[0058] In an embodiment, the delivery-enhancing agent is included in thebuffer in which the recombinant adenoviral vector delivery system isformulated. The delivery-enhancing agent may be administered prior tothe recombinant virus or concomitant with the virus. In someembodiments, the delivery-enhancing agent is provided with the virus bymixing a virus preparation with a delivery-enhancing agent formulationjust prior to administration to the patient. In other embodiments, thedelivery-enhancing agent and virus are provided in a single vial to thecaregiver for administration.

[0059] In the case of a pharmaceutical composition comprising a tumorsuppressor gene contained in a recombinant adenoviral vector deliverysystem formulated in a buffer which further comprises adelivery-enhancing agent, the pharmaceutical composition may beadministered over time in the range of about 5 minutes to 3 hours,preferably about 10 minutes to 120 minutes, and preferably about 15minutes to 90 minutes. In another embodiment the delivery-enhancingagent may be administered prior to administration of the recombinantadenoviral vector delivery system containing the tumor suppressor gene.The prior administration of the delivery-enhancing agent may be in therange of about 30 seconds to 1 hour, preferably about 1 minute to 10minutes, and preferably about 1 minute to 5 minutes prior toadministration of the adenoviral vector delivery system containing thetumor suppressor gene.

[0060] Solvents that may be used for the formulations of the presentinvention include, for example, aqueous solvents such as water forinjection, and/or nonaqueous solvents, such as DMSO andN,N-Dimethyylacetamide, also known as DMA, and co-solvent mixtures,e.g., glycerol and water, as prepared preferably in accordance with USPstandards.

[0061] The formulations preferably contain polysorbates, orpolyoxyethylene sorbitan esters, a class of nonionic surfactants thatincluded, e.g., polysorbate 80 and polysorbate 20, amongst others,Pluronics, or polyethylenepolypropylene glycol block copolymers, a classof nonionic surfactants, that include e.g. Pluronic L68 and L92, amongstothers, and hydroxypropyl-beta-cyclodextrin, a polysubstitutedhydroxyalkyl-beta-cyclodextrin, which is a class of nonionic complexingagents, that include, e.g., HPβCD and BigCHAP. Preferred are HPβCD,BigCHAP, Polysorbate 80, Polysorbate 20, Pluronic L64, and Pluronic L92as solubilizing agents. The solubilizers can be used, for example,either individually or in combinations. The concentrations of thesolubilizing agents are set forth below. HPβCD can be present in aconcentration of about 50 to 500 mg/ml, BigCHAP can be present in aconcentration of about 20 to about 360 mg/ml, Polysorbate 80 can bepresent in a concentration of about 1 to 36 mg/ml, the Pluronics can bepresent in concentrations of about 1 to about 150 mg/ml, and the otheringredients may be present in concentrations as set forth below.

[0062] The concentration of the delivery-enhancing agent will depend ona number of factors known to one of ordinary skill in the art such asthe particular delivery-enhancing agent being used, the buffer, pH,target tissue or organ and mode of administration. The concentration ofthe delivery-enhancing agent will be in the range of 0.01% to 50% (w/v),preferably 10% to 40% (w/v), preferably 14% to 19% (w/v), and preferably0.01% to 30% (w/v). Preferably, the detergent concentration will beabout 1% to 12% (w/v) in the formulation prior to admixture, andpreferably 0.1% (w/v) of the formulation when in admixture. Preferably,the detergent concentration in the final formulation administered to thepatient is about 0.5-2 times the critical micellization concentration(CMC). The CMC is equal to 0.001 mg/ml in the recombinant adenovirusformulation.

[0063] The lyophilized formulations of SYN3 preferably contain a citratebuffering system. More preferably, the citrate buffering system cancomprise at least one citric buffer, such as citric acid monohydrate USPor sodium citrate dihydrate USP. More preferably, the citrate bufferingsystem comprises a combination of citric acid monohydrate USP and sodiumcitrate dihydrate USP. When used in combination, the amount of citricacid monohydrate USP can be present in a concentration of about 0.005 toabout 2 mg/ml, more preferably 0.016 to about 1.35 mg/ml, preferably0.016 to about 0.72 mg/ml, preferably about 0.005 to about 1.35, and thesodium citrate dihydrate USP can be present in a concentration of about0.02 to about 5.37 mg/ml, preferably 0.05 to 3.00 mg/ml, preferably 0.05to 2.31 mg/ml. Other suitable buffering systems that are suitableinclude, for example, phosphate, glycine, either in place of the citratebuffering system or in combination therewith, and varying combinationsof all of the above.

[0064] The buffering system will provide a pH of the lyophilizedformulation such that there is improved stability. Preferably, the pHwill be in a range of about 5 to about 6. The admixture aqueousformulations of SYN3 are preferably buffered at about a pH of about 7 toabout 8.5, preferably about 7.4, and SYN3 remains stable in thedehydrated powder for at least 3 months at 40° C.

[0065] The lyophilized formulations preferably contain glycine ormannitol as freeze-drying bulking agents. Other suitable freeze-dryingbulking agents that may be used include, for example, lactose,recombinant gelatin, and methylcellulose. The freeze drying-bulkingagent may be present in a concentration of from about 5 to 100 mg/mlwhen the agent is mannitol, and about 10 to 200 mg/ml when the agent isglycine.

[0066] The lyophilized formulations preferably contain ascorbic acid asan antioxidant. Other suitable antioxidants that may be used include,for example, citric acid. When ascorbic acids is the antioxidant, it maybe present in a concentration of about 0.001 to about 0.6 mg/ml.

[0067] The compositions of this invention may additionally include, forexample, a stabilizer, enhancer or other pharmaceutically acceptablecarriers or vehicles. A pharmaceutically acceptable carrier can containa physiologically acceptable compound that acts, for example, tostabilize the recombinant adenoviral vector delivery system comprisingthe tumor suppressor gene A physiologically acceptable compound caninclude, for example, carbohydrates, such as glucose, sucrose ordextrans, Hydroxypropyl-β-Cyclodextrin, antioxidants, such as ascorbicacid or glutathione, chelating agents, low molecular weight proteins orother stabilizers or excipients.

[0068] Other physiologically acceptable compounds include, for example,wetting agents, emulsifying agents, dispersing agents or preservatives,which are particularly useful for preventing the growth or action ofmicroorganisms. Various preservatives are well known and include, forexample, phenol and ascorbic acid. One skilled in the art would knowthat the choice of pharmaceutically acceptable carrier, depends on theroute of administration and the particular physiochemicalcharacteristics of the recombinant adenoviral vector delivery system andthe particular tumor suppressor gene contained therein. Examples ofcarriers, stabilizers or adjuvants can be found in Gennaro, Remington's:The Science and Practice of Pharmacy, 19th Ed. (Mack Publishing. Co.,Easton, Pa. 1995), incorporated herein by reference.

[0069] The recombinant viral vector delivery system comprising atherapeutic gene formulated in a buffer comprising a delivery-enhancingagent may be delivered to any cancerous tissue or organ using anydelivery method known to the ordinarily skilled artisan for example,intratumoral or intravesical administration. Cancerous tissues andorgans include, for example, any tissue or organ having an epithelialmembrane such as the gastrointestinal tract, the bladder, respiratorytract, and the lung. Examples include but are not limited to carcinomaof the bladder and upper respiratory tract, vulva, cervix, vagina orbronchi; local metastatic tumors of the peritoneum; broncho-alveolarcarcinoma; pleural metastatic carcinoma; carcinoma of the mouth andtonsils; carcinoma of the nasopharynx, nose, larynx, oesophagus,stomach, colon and rectum, gallbladder, or skin; or melanoma.

[0070] The delivery-enhancing agents of the invention can also be usedto formulate other pharmaceutical agents, such as proteins, nucleicacids, antisense RNA, small molecules, etc., for administration to anytissue or organ having an epithelial membrane.

[0071] Administration of Formulations

[0072] In some embodiments, the delivery-enhancing agent is included inthe buffer in which an expression vector is formulated. Thedelivery-enhancing agent can be administered prior to the expressionvector or concomitant with the expression vector. In some embodiments,the delivery-enhancing agent is provided with the expression vector bymixing an expression vector with a delivery-enhancing agent formulationjust prior to administration to the patient. In other embodiments, thedelivery-enhancing agent and the expression vector are provided in asingle vial to the caregiver for administration.

[0073] In the case of a pharmaceutical composition comprising a tumorsuppressor gene contained in a recombinant adenoviral vector deliverysystem formulated in a buffer which further comprises adelivery-enhancing agent, the pharmaceutical composition can beadministered over time in the range of about 5 minutes to 3 hours,preferably about 10 minutes to 120 minutes, and most preferably about 15minutes to 90 minutes. In another embodiment the delivery-enhancingagent may be administered prior to administration of the recombinantadenoviral vector delivery system containing the tumor suppressor gene.The prior administration of the delivery-enhancing agent may be in therange of about 30 seconds to 1 hour, preferably about 1 minute to 10minutes, and most preferably about 1 minute to 5 minutes prior toadministration of the adenoviral vector delivery system containing thetumor suppressor gene.

[0074] The expression vector formulated in a buffer comprising adelivery-enhancing agent can be delivered to any tissue or organ,including neoplastic tissues such as cancer tissue, using any deliverymethod known to the ordinarily skilled artisan for example, intratumoralor intravesical administration. Tissues and organs include any tissue ororgan having an epithelial membrane such as the gastrointestinal tract,the bladder, respiratory tract, and the lung. Examples include but arenot limited to carcinoma of the bladder and upper respiratory tract,vulva, cervix, vagina or bronchi; local metastatic tumors of theperitoneum; broncho-alveolar carcinoma; pleural metastatic carcinoma;carcinoma of the mouth and tonsils; carcinoma of the nasopharynx, nose,larynx, oesophagus, stomach, colon and rectum, gallbladder, or skin; ormelanoma.

[0075] In some embodiments of the invention, an expression vector isformulated in mucosal, topical, and/or buccal formulations, particularlymucoadhesive gel and topical gel formulations. Exemplary permeationenhancing compositions, polymer matrices, and mucoadhesive gelpreparations for transdermal delivery are disclosed in U.S. Pat. No.5,346,701. Such formulations are especially useful for the treatment ofcancers of the mouth, head and neck cancers (e.g., cancers of thetracheobronchial epithelium) skin cancers (e.g., melanoma, basal andsquamous cell carcinomas), cancers of the intestinal mucosa, vaginalmucosa, and cervical cancer.

[0076] In some embodiments of the invention, a therapeutic agent isformulated in ophthalmic formulations for administration to the eye.Such formulations are useful in the delivery of the retinoblastoma (RB)gene to the eye, optionally in conjunction with the delivery of p53.

[0077] Methods of Treatment

[0078] The composition of the invention are typically administered toenhance transfer of gene to a cell. The cell can be provided as part ofa tissue, such as an epithelial membrane, or as an isolated cell, suchas in tissue culture. The cell can be provided in vivo, ex vivo, or invitro.

[0079] The compositions can be introduced into the tissue of interest invivo or ex vivo by a variety of methods. In some embodiments of theinvention, the modulating agent is introduced to cells by such methodsas microinjection, calcium phosphate precipitation, liposome fusion, orbiolistics. In further embodiments, the therapeutic agent is taken updirectly by the tissue of interest.

[0080] In some embodiments of the invention, the compositions of theinvention are administered ex vivo to cells or tissues explanted from apatient, then returned to the patient. Examples of ex vivoadministration of therapeutic gene constructs include Arteaga et al.,Cancer Research 56(5):1098-1103 (1996); Nolta et al., Proc Natl. Acad.Sci. USA 93(6):2414-9 (1996); Koc et al., Seminars in Oncology 23(1):46-65 (1996); Raper et al., Annals of Surgery 223(2):116-26 (1996);Dalesandro et al., J. Thorac. Cardi. Surg., 11(2):416-22 (1996); andMakarov et al., Proc. Natl. Acad. Sci. USA 93(1):402-6 (1996).

EXAMPLES

[0081] The following examples will further illustrate the presentinvention.

Example 1

[0082] The following table represents ranges of the ingredients fornonaqueous liquid formulations of the present invention. Prior toadministration (for bladder cancer), the SYN3 solution is combined withthe recombinant adenovirus preparation in a 1:50 v/v ratio to form anadmixture that is administered to the patient. Range of IngredientConcentrations Ingredient mg/ml mg/ml mg/ml mg/ml SYN3 0.001-1500.001-150 0.001-150 0.001-150 Polysorbate 20 0.001-150 Polysorbate 800.001-150 Pluronic L64 0.001-100 Pluronic L92 0.001-100N,N-Dimethylacetamide 1 ml 1 ml 1 ml 1 ml qs ad

[0083] To prepare, weigh approximately 75% of DMA into a glass beaker.To a separate beaker, charge the surfactant (Polysorbate 80, Polysorbate20, Pluronic L64 or Pluronic L92) and dissolve in a small volume(approximately 10% of final volume) of DMA. Charge the DMA/surfactantsolution into the DMA with constant stirring. Preweigh SYN3 in aseparate container. Slowly charge the SYN3 into the solution whilestirring. Once the SYN3 is dissolved, add sufficient DMA to final volumeby weight (density=0.962 g/ml at 25° C.). Filter the solution through a0.22 filter attached to a syringe equipped with a nonlatex plunger andstore the solution in a tightly sealed glass container at 4° C.

[0084] The following Examples may be prepared in accordance with Example1.

Example 2

[0085] Ingredient mg/ml SYN3 51 Polysorbate 20 50 N,N-Dimethylacetamideqs ad 1 ml

Example 3

[0086] Ingredient mg/ml SYN3 51 Polysorbate 80 50 N,N-Dimethylacetamideqs ad 1 ml

Example 4

[0087] Ingredient mg/ml SYN3 51 Pluronic L64 25 N,N-Dimethylacetamide qsad 1 ml

Example 5

[0088] Ingredient mg/ml SYN3 51 Pluronic L92 25 N,N-Dimethylacetamide qsad 1 ml

[0089] The following 25 ml batches were prepared according to the sameprocedure.

Example 6

[0090] Ingredient mg/ml SYN3 1.275 Polysorbate 20 1.250N,N-Dimethylacetamide qs ad 25 ml

Example 7

[0091] Ingredient mg/ml SYN3 1.275 Polysorbate 80 1.250N,N-Dimethylacetamide qs ad 25 ml

Example 8

[0092] Ingredient mg/ml SYN3 1.275 Pluronic L64 0.625N,N-Dimethylacetamide qs ad 25 ml

Example 9

[0093] Ingredient mg/ml SYN3 1.275 Pluronic L92 0.625N,N-Dimethylacetamide qs ad 25 ml

[0094] The following is a stability analysis of Examples 2, 3, 4, and 5.

Example 10

[0095] T° −20° C. 4° C. 25° C. 55° C. −80° C. Ex. No. 2 1 week 52.2252.15 52.38 51.36 52.31 2 week 52.16 52.14 52.38 52.68 3 week 51.9252.38 51.98 51.46 4 week 52.1 52.63 52.28 51.15 Ex. No. 3 1 week 51.953.75 53.09 52.98 53.33 2 week 53.46 52.9 51.98 51.75 3 week 52.64 53.3553.41 50.6 4 week 53.41 52.96 53.85 55.81 Ex. No. 4 1 week 51.34 53.9453.68 53.76 53.98 2 week 53.3 53.43 53.76 52.23 3 week 53.14 52.56 52.354 week 53.72 53.67 54.2 53.42 Ex. No. 5 1 week 52.34 52.16 52.43 51.4852.33 2 week 51.64 51.5 51.39 52.18 3 week 52.28 52.56 53.73 53.01 4week 53.49 53.49 53.67 53.73

[0096] Stability testing was accomplished by HPLC. Solution formulationswere placed in the indicated temperature conditions, incubated forspecified times and concentrations were determined by HPLC and comparedto initial concentrations (−80° C.). The nonaqueous, solutionformulations of SYN3 remain stable for at least 1 month at 55° C. whenSYN3 is dissolved in N,N-Dimethylacetamide (DMA).

Example 11

[0097] The following table represents ranges of the ingredients forlyophilized formulations of the present invention. The compoundedsolution is filled as indicated into a 20-ml capacity Type II glass vialand lyophilized. Preparation for administration requires addition of 20ml of WFI to the vial containing the freeze-dried cake to redissolve theSYN3. The SYN3 solution is combined with p53, or any recombinantadenovirus preparation, in a v/v ratio of 1:5. The admixture is thenadministered to the patient for, for instance, bladder cancer. Range ofConcentrations INGREDIENT mg/ml mg/ml mg/ml SYN3 0.001-150 0.001-1500.001-150 Citric Acid Monohydrate 0.016-0.72 0.016-0.96 0.005-1.35Sodium Citrate Dihydrate  0.05-2.31  0.05-3  0.02-5.37Hydroxypropyl-β-cyclodextrin —   50-500 — BigCHAP   20-360 — — Glycine  10-200 — — Mannitol — —    5-100 Polysorbate 80 —  1.0-36.0   10-200Ascorbic Acid^(a) 0.001-0.6 0.001-0.6 — Water for Injection (WFI) qs  1ml  1 ml  1 ml ad PH Range    5-6    5-6    5-6 ml Fill into 20-mlvial^(b) 5.3 5.3 5.3 Reconstitution Volume 20 ml 20 ml 20 ml of WFI^(c)

[0098] The following examples are methods of preparing the lyophilizedformulations.

Example 12

[0099] Ingredient Grams/Liter SYN3 24 Citric Acid Monohydrate USP 0.24Sodium Citrate Dihydrate USP 0.77 Big CHAP 120 Glycine USP 50 AscorbicAcid USP 0.25 Water for Injection USP qs ad 1000 ml

[0100] The actual amount of SYN3 to be charged will be adjustedaccording to the purity of the drug substance batch using the followingformula:

grams SYN3=24.0×100/(% Purity).

[0101] For example:

[0102] SYN3 drug substance=97.0% pure.

[0103] 24.0×100/97.0=24.7 grams SYN3 to be charged for a 1-Liter batch.

[0104] Accordingly, to determine the amount of SYN3 that will be chargedto the batch according to the following formula:

g SYN3/Liter=24.0 g/Liter×[100/(% SYN3 Batch Purity)]

[0105] The volume of Water for Injection to be charged to the batch isto be determined according to the following formula:

Volume of Water for Injection (Liters)=Batch Volume (Liters)×0.5

[0106] Charge the volume of water for injection calculated using theformula above into a tared compounding vessel equipped with an agitator.Charge and dissolve, with agitation, the BigCHAP. Sterile Water forInjection may be used to rinse the weighing vessel to retrieve all ofthe material. Complete dissolution of BigCHAP may require approximately30 to 60 minutes of continuous agitation at a moderate stirring rate.Charge and dissolve with agitation (moderate stirring rate) the SYN3into the BigCHAP solution. Water for Injection may be used to rinse theweighing vessel to retrieve all of the material. Complete dissolution ofSYN3 may require up to 1 hour of mixing. Charge and dissolve withagitation and in order: Glycine, Ascorbic Acid, Citric Acid Monohydrateand Sodium Citrate Dihydrate into the solution that contains both BigCHAP and SYN3. Water for Injection may be used to rinse the weighingvessels to retrieve all of the material. Add Water for Injection tobring the batch to the final volume (density of the solution isapproximately 1.05 1 g/ml at 25° C.). Mix the solution for a minimum of15 minutes.

[0107] Remove a small (<5 ml) sample of the solution for pH measurement.The pH should be between 5.0 and 6.0. No pH adjustment is necessary. Oneof ordinary skill in the art can readily ascertain the pH of theresulting product.

[0108] To complete compounding, aseptically filter the solution. Ifnecessary, the compounded batch may be stored at 2° C. to 8° C. for upto 24 hours in a sealed, sterilized, stainless steel pressure vesselprior to filling into the vials. The batch may be filtered more thanonce to assure sterility.

[0109] Aseptically fill 5.3±0.1 ml of solution into 20-ml Type 1 flintglass vials that have been washed and sterilized. Aseptically insert20-mm West 4416/50 lyo-shape rubber stoppers that have been washed,siliconized and sterilized into the vials in the lyophilizationposition.

[0110] Precool the lyophilizer shelves to 4±2° C. Aseptically load thetrays of filled vials onto the lyophilizer shelves. After all the traysare loaded, cool the shelves to −40° C. in 1 hour and maintain theproduct at −35° C. or below for at least 4 hours before proceeding.Start cooling the condenser. When the condenser temperature is at −45°C. or below, begin evacuating the chamber. When 50-70 mm Hg of vacuumpressure is attained, heat the shelf temperature to −20° C. over 0.5hour. Maintain the shelf temperature at −20° C. for 36 hours atapproximately 150 mm Hg pressure (100 to 200 mm Hg pressure). Producttemperature must remain at or above −20° C. for at least 6 hours beforeproceeding. Heat the shelf to 25° C. in 1 hour and reduce pressure toapproximately 50 mm Hg pressure. Maintain the shelf temperature at 25°C. at approximately 50 mm Hg pressure for 14 hours. Vent the chamberwith sterile filtered nitrogen to approximately 950 mm Hg. Stopper thevials inside the lyophilizer. Remove the vials from the lyophilizer andcrimp the vials with 20-mm aluminum seals. The vials should be stored at2° C. to 8° C. until inspection is completed.

[0111] The product is a white to off-white cake. The vials should bestored between 2° C. to 8° C. after inspection. For labeling andinspection purposes, the vials may be exposed to 19° C.-25° C. for up to6 hours.

[0112] The following examples were prepared in accordance with the batchpreparation as set forth in Example 12 above.

Example 13

[0113] Ingredient mg/ml SYN3 24 Citric Acid Monohydrate USP 0.24 SodiumCitrate Dihydrate USP 0.77 Big CHAP 120 Glycine USP 50 Ascorbic Acid USP0.25 Water for Injection USP qs ad 1 Ml

[0114] The pH of the resulting product was 5.34. Ingredient mg/ml SYN324 Citric Acid Monohydrate USP 0.32 Sodium Citrate Dihydrate USP 1.02HPβCD 200 Polysorbate 80 12 Ascorbic Acid USP 0.25 Water for InjectionUSP qs ad 1 ml

[0115] The pH of the resulting product was 5.45.

Example 15

[0116] Ingredient mg/ml SYN3 24 Citric Acid Monohydrate USP 0.45 SodiumCitrate Dihydrate USP 1.79 Mannitol 30 Polysorbate 80 72 Water forInjection USP qs ad 1000 ml

[0117] The pH of the resulting product was 5.76.

[0118] Stability testing of the resultant lyophilized products ofExamples 13, 14 and 15 yielded the following results:

Example 16

[0119] EXAMPLES 13 14 15 CONDITION mg/ml mg/ml mg/ml Initial 6.05 5.896.06  2 weeks −20° C. 6.02 6.04 6.28  2 weeks 4° C. 6.17 5.98 6.17  2weeks 25° C. 6.14 5.91 6.62  2 weeks 40° C. 5.87 5.78 6.32  4 weeks −20°C. 6.81 5.71 6.11  4 weeks 4° C. 6.75 6.63 6.75  4 weeks 25° C. 6.816.40 6.92  4 weeks 40° C. 6.73 6.66 6.58 12 weeks 4° C. 6.74 6.66 6.7412 weeks 25° C. 6.74 6.45 6.72 12 weeks 40° C. 6.61 6.52 6.71

[0120] Stability testing was accomplished by HPLC. Lyophilizedformulations were reconstituted (redissolved) with 19.5 ml WFI. Sampleswere placed in the indicated temperature conditions, incubated forspecified times and concentrations were determined by HPLC and comparedto initial concentrations.

Example 17

[0121] Ingredient mg/ml SYN3 24.0 Citric Acid Monohydrate USP 0.32Sodium Citrate Dihydrate USP 1.02 Hydroxypropyl-β-cyclodextrin 200Polysorbate 80 12.0 Ascorbic Acid USP 0.25 Water for Injection USP qs ad1000 ml

[0122] The actual amount of SYN3 to be charged will be adjustedaccording to the purity of the drug substance batch using the followingformula:

grams SYN3=24.0×100/(% Purity).

[0123] Sample Calculation:

[0124] SYN3 drug substance=97.0% pure.

[0125] 24.0×100/97.0=24.7 grams SYN3 to be charged for a 1-Liter batch.

[0126] The following Example was prepared as such: Initially, determinethe amount of SYN3 that will be charged to the batch according to thefollowing formula:

g SYN3/Liter=24.0 g/Liter×[100/(% SYN3 Batch Purity)]

[0127] Next, determine the volume of Water for Injection to be chargedto the batch according to the following formula:

Volume of Water for Injection (Liters)=Batch Volume (Liters)×0.5

[0128] Charge the volume of Water for Injection into a tared compoundingvessel equipped with an agitator. Charge and dissolve, with agitation,the Hydroxypropyl-β-cyclodextrin. Note, complete dissolution ofHydroxypropyl-β-cyclodextrin may require approximately 30 to 45 minutesof continuous agitation at a moderate stirring speed. Water forInjection may be used to rinse weighing vessel to retrieve all of thematerial. Charge and dissolve the Polysorbate 80 to the solution. ThePolysorbate 80 may be predissolved in 0.1× total batch volume of Waterfor Injection (Liters) and charged to the solution.

[0129] Charge and dissolve with agitation the SYN3 into the solution.Complete dissolution of SYN3 may require up to 1 hour of mixing. Waterfor Injection may be used to rinse weighing vessel to retrieve all ofthe material.

[0130] Charge and dissolve with agitation and in order: Ascorbic Acid,Citric Acid monohydrate and Sodium Citrate dihydrate into the solution.Water for Injection may be used to rinse weighing vessels to retrieveall of the material. Add the Water for Injection to bring the batch tothe final volume (density of solution is 1.058 g/ml at 25° C.). Mix thesolution for a minimum of 15 minutes.

[0131] Remove a small (<5 ml) sample of the solution for pH measurement.The pH should be between 5.0 and 6.0. No pH adjustment is necessary.Aspetically filter the solution that has been washed and tested forintegrity into a sterilized, stainless steel pressure vessel orequivalent should be used. If necessary, the compounded batch may bestored at 2° C. to 8° C. for up to 24 hours in a sealed, sterilized,stainless steel pressure vessel prior to filling. The batch may befiltered more than once to assure sterility.

[0132] Aseptically fill 5.3±0.1 ml of solution into 20-ml Type 1 flintglass vials that have been washed and sterilized. Aseptically insertlyo-shape rubber that have been washed, siliconized and sterilized intothe vials in the lyophilization position.

[0133] To lyophilize, precool the lyophilizer shelves to 4±2° C.Aseptically load the trays of filled vials onto the lyophilizer shelves.After all the trays are loaded, cool the shelves to −40° C. in 1 hourand maintain the product at −35° C. or below for at least 4 hours beforeproceeding. Start cooling the condenser. When the condenser temperatureis at −45° C. or below, begin evacuating the chamber. When 50-70 mm Hgof vacuum pressure is attained, heat the shelf temperature to −20° C.over 0.5 hour. Maintain the shelf temperature at −20° C. for 36 hours atapproximately 150 mm Hg pressure (100 to 200 mm Hg pressure). Producttemperature must remain at or above −20° C. for at least 6 hours beforeproceeding. Heat the shelf to 25° C. in 1 hour and reduce pressure toapproximately 50 mm Hg pressure. Maintain the shelf temperature at 25°C. at approximately 50 mm Hg pressure for 14 hours. Vent the chamberwith sterile filtered nitrogen to approximately 950 mm Hg. Stopper thevials inside the lyophilizer. Remove the vials from the lyophilizer andcrimp the vials with 20-mm aluminum. The vials should be stored at 2° C.to 8° C. until inspection is completed.

[0134] The product is a white to off-white cake. The vials should bestored between 2° C. to 8° C. after inspection. For labeling andinspection purposes, the vials may be exposed to 19° C.-25° C. for up to6 hours.

[0135] p53 (rAD/p53) remains stable when combined with the lyophilizedformulations of SYN3 for at least 2 hours at 37° C. and 24 hours at 25°C. p53 remains stable when combined with the aqueous solutionformulations of SYN3 for at least 4 hours at 37° C. and 24 hours at 25°C.

Example 18

[0136] This example illustrates the synthesis of SYN3.

[0137] Part 1: Synthesis of Compound III

[0138] The synthetic scheme for SYN3 is shown in FIG. 2, which isadapted from U.S. Pat. No. 6,392,069. The lactone of lactobionic acid(II) was synthesized by dissolving one g (2.8 mmol) of lactobionic acid(I) in 50 ml of methanol, evaporating to dryness on a rotary evaporator,and repeating this process six times. To obtain Compound III, theresulting residue (II) was dissolved in 50 ml of isopropanol by heatingto 50° C. To this solution was added 1.2 ml (8.4 mmol) ofN-3-aminopropyl)-1,2-propanediamene. The temperature was increased to100° C., and the solution was stirred for three hours. The solvent wasremoved by rotary evaporation and the resulting residue was washedseveral times with chloroform to remove excess unreactedN-(3-aminopropyl)-1,3-propanediamene. The remaining residue (III) wasused as is in Part 3 below.

[0139] Part 2: Synthesis of Compound IV

[0140] Compound IV was synthesized by dissolving 2.28 g of cholic acid(5.6 mmol) in N,N-dimethylformamide by heating to 60° C. Triethylamine(0.78 ml (5.6 mmol)) was added and the solution was cooled in an icebath. Isobutyl chloroformate (0.73 ml (5.6 mmol)) was then added and awhite precipitate formed as the stirring was continued for ten minutes.

[0141] Part 3: Synthesis of SYN3 (Compound V)

[0142] Compound III was dissolved in N, N-dimethylformamide, cooled inan ice bath, and stirred. The suspension resulting from the synthesis ofCompound IV was filtered into the solution containing Compound III. Theresulting solution was stirred at room temperature for 6 hrs. Thesolvent was removed using high vacuum rotary evaporation and the residuewas dissolved in 100 ml of chloroform/methanol (50/50). Twenty-five mlof this solution was purified by silica gel flash chromatography usingchloroform/methanol (60/40) as the elution solvent. Analysis of thefractions eluting from the column was conducted by silica gel thin layerchromatography using a mobile phase consisting ofchloroform/methanol/water/concentrated ammonium hydroxide (100/80/10/5).The compounds were visualized by charring after spraying with ethanolicsulfuric acid. Fractions containing product were consolidated andrepurified using flash chromatography andchloroform/methanol/water/concentrated ammonium hydroxide (100/80/10/5)as the elution solvent. Fractions containing product were consolidatedand evaporated to a white powder (300 mg of Compound V). ¹H-NMR andMALDI mass spectrometric analysis of the product were consistent withthe structure shown.

[0143] As will be apparent to those skilled in the art to which theinvention pertains, the present invention may be embodied in forms otherthan those specifically disclosed above, without departing from thespirit or essential characteristics of the invention. The particularembodiments of the invention described above, are, therefore to beconsidered as illustrative and not restrictive. The scope of the presentinvention is as set forth in the appended claims rather than beinglimited to the Examples contained in the foregoing description.

[0144] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications andpatent applications cited in this specification are herein incorporatedby reference in their entirety for all purposes as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

What is claimed is:
 1. A pharmaceutical composition comprising SYN3 incombination with a pharmaceutically acceptable carrier.
 2. Thepharmaceutical composition of claim 1, wherein said composition furthercomprises at least one pharmaceutically acceptable solubilizer.
 3. Thepharmaceutical composition of claim 2, wherein said composition islyophilized.
 4. The lyophilized pharmaceutical composition of claim 3,wherein said composition further comprises at least one pharmaceuticallyacceptable bulking agent.
 5. The pharmaceutical composition of claim 1,wherein said composition further comprises an expression vectorcomprising a foreign DNA sequence inserted into the vector.
 6. Thepharmaceutical composition of claim 5, wherein said composition furthercomprises at least one pharmaceutically acceptable solubilizer.
 7. Thepharmaceutical composition of claim 1, wherein said pharmaceuticallyacceptable carrier is a nonaqueous carrier.
 8. The pharmaceuticalcomposition of claim 7, wherein said composition further comprises atleast one pharmaceutically acceptable solubilizer.
 9. The pharmaceuticalcomposition of claim 7, wherein said composition further comprises anexpression vector comprising a foreign DNA sequence inserted into thevector.
 10. The pharmaceutical composition of claim 9, wherein saidcomposition further comprises at least one pharmaceutically acceptablesolubilizer.
 11. The pharmaceutical composition of claim 1, wherein saidpharmaceutically acceptable carrier is an aqueous carrier.
 12. Thepharmaceutical composition of claim 11, wherein said composition furthercomprises at least one pharmaceutically acceptable solubilizer.
 13. Thepharmaceutical composition of claim 12, wherein said composition furthercomprises at least one pharmaceutically acceptable bulking agent. 14.The pharmaceutical composition of claim 13, wherein said compositionfurther comprises at least one pharmaceutically acceptable bufferingagent.
 15. The pharmaceutical composition of claim 11, wherein saidcomposition further comprises an expression vector comprising a foreignDNA sequence inserted into the vector.
 16. The pharmaceuticalcomposition of claim 15, wherein said composition further comprises atleast one pharmaceutically acceptable solubilizer.
 17. Thepharmaceutical composition of claim 16, wherein said composition furthercomprises at least one pharmaceutically acceptable bulking agent. 18.The pharmaceutical composition of claim 17, wherein said compositionfurther comprises at least one pharmaceutically acceptable bufferingagent.
 19. A method of treating a disease in a mammal, said methodcomprising administering a therapeutically effective amount of apharmaceutical composition comprising SYN3 in combination with apharmaceutically acceptable carrier.